Vol. 166, No. 2, 1990 January 30, 1990

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AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 984-992

cDNA CLONING OF A NOVEL CELL ADHESION PROTEIN EXPRESSED IN HUMAN SQUAMOUS CARCINOMA CELLS Yuk-Chor Wong, *Sai-Wah Tsao, Mika Kakefuda and Samuel D. Bernal Section of Medical Oncology, Boston University Medical Center, 75 E. Newton Street, Boston, MA 02118 Received

December

11,

1989

A novel protein (SQMl protein) present in human squamous epithelial cells has been found to be involved in cell adhesion in squamous epithelial cells, endothelial cells and extracellular matrix proteins. The corresponding cDNA that encodes a 135residue poly peptide has been isolated. Sequence analysis indicates that the encoded polypeptide is distinct yet related to the p subunit of integrins. A new sequence motif consisting of a heptadic repeat of positively-charged residues present in the polypeptide has been identified and is proposed to be important in protein-protein interaction. These results suggest that SQMl protein, a new cell adhesion molecule expressed in squamous epithelial cells, may play an important role in tumor metastasis. 0 1990 Academic Press, Inc.

Cell adhesion molecules which determine specificity of cell-cell and cell-substrate interactions are thought to be important in tumor growth, tumor metastasis, embryonic development, cellular differentiation, immunological response, wound healing and coagulation (l-3). Several membrane molecules which appear to participate in thesevariousprocesses have recently been described. These include the family of integrins, which are a diverse group of transmembrane glycoproteins made up of heterodimers of o and /3 subunits (4). Integrins are known to interact with other cells, ECM glycoproteins, complements and clotting factors while their intracellular domains interact with cytoskeleton (4). Other cell adhesion molecules include molecules involved in lymphocyte, leukocyte and platelet adhesion (3). Intercellular adhesion molecules such as N-CAM and LCAM are also known to be important in morphogenesis during embryonic development (2). In contrast to the extensive data on immune and endothelial cells, little information is available on epithelial cell adhesion molecules. Recently, we have identified a new protein (SQMl protein) which appears to play a role in adhesion of human carcinoma cells to endothelial cells and to ECM proteins. This protein is recognized by monoclonal antibody * Present address: Department of Pathology, Dana-Farber Cancer Institute and Harvard Medical School, 44 BiMey St., Boston, MA 02115. . . B: ECM, extracellular matrix, vWF, von Willebrand factor; GpIIb, glycoprotein IIb; GpIIIa, glycoprotein IIIa. 0006-291X/90 Copyright All rights

$1.50

0 1990 by Academic Press, Inc. of reproduction in any form reserved.

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SQMl which we developed against the surface membrane

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of human squamous carcinomas

($6). In this report, we describe the inhibitive effect of SQMl antibody on cell adhesion of squamous epithelial cells, endothelial cells and ECM proteins. We have also cloned and sequenced the cDNA of SQMl protein. Results of sequence analysis seem to suggest that SQMl protein is distinct yet related to the p subunit of integrins. MATERIALS

AND METHODS

The human cell lines: squamous carcinoma SCC25 was from Dr. J. G. Rheinwald; squamous carcinoma Calu-1 and endothelial HUVEC were from American Type Culture Collection; lymphoblastic leukemia CEM was from Dr. H. Lazarus. Fibronectin and collagen were from Sigma Chem. Co.(St. Louis, MO). Rhodaminewas from Eastman Organic Chem. (Rochester, NY). Enzymes, linker and the rabbit reticulocyte system were from BRL (Bethesda, MD). GeneScreen was from NEN (Boston, MA) and Ml3 dideoxy sequencing kit from Pharmacia (Piscataway, NJ). Cell Cells were grown in RPM1 1640 medium supplemented with 10% -- Culture. calf bovine serum, 2 mM 1-glutamine, 100 mu/ml penicillin and 100 ug/ml streptomycin at 370C in a 5 % CO2 atmosphere. Cell Adhesion Studies: The test cells were preincubated with SQMl antibody for 1 hour. As controls, similar protein concentration of normal serum or an irrelevant antibody was used instead of SQMl antibody. After washing with medium, the cells were labelled with a fluorescent dye, Rh-123, for 30 min (7) and then washed with medium. The labelled cells were added to round coverslips with cell monolayers or ECM proteins. After 2 hrs, the coverslips were rinsed with medium and examined by fluorescence microscopy to count the total number of adherent cells. In Vitro Translation and Immunoblot Analvsis: In vitro translation of 5 ug of SCC25 total mRNA or 0.5 ug of rabbit globin mRNA using the rabbit reticulocyte system was performed as recommended by the manufacturer. The translation products were run on a 13% SDS-polyacrylamide gel and electroblotted and indirectly immunostained as previously described (8). cDNALibrarv Co struct o a d S e ’ P: Total RNA was isolated from cells b the guanidinium/lithium cnhloridi ~e~hodcr(~oly(A)+RNA was prepared by oligo (’dT)cellulose chromatography. Double-stranded cDNA was synthesized as previously described (10) and internal EcoRl sites were protected with methylation. The cDNA was then ligated to dephosphorylated EcoRl digested lambda gtll arms via EcoRl linkers. The cDNA library was then packaged in vitro! titered and amplified in Y1090 E. coli. Aliquots of the library were plated out for screening with SQMl antibody (11). The secondary antibody used was goat anti-mouse IgM alkaline phosphatase conjugate. Candidate clones were isolated and then secondary and tertiary screened. The insert of the lambda gtll cDNA : clones was excised by EcoRl digestion, and cloned into Ml3 m 18. The DNA sequence was determined by the dideoxynucleotide chain termination metho cr(12). Sequence analysis was done using programs supplied by the Molecular Biology Computer Research Resource of the Dana-Farber Cancer Institute. Northern Blot Analysis: Total RNA (10 ug/lane) was electrophoresed on a 1.2 % agarose-formaldehyde gel and blotted onto GeneScreen. Either nick-translated MWRF or labeled anti-sense strand derived from a Ml3 subclone of a lambda clone, Q12 was used as probe. RESULTS AND DISCUSSION

Effect of SOMl Antibodv on Cell Adhesion: To assay the possible adhesive function of SQMl protein, we measured the effect of SQMl monoclonal antibody on cell-cell adhesion, compared to control antibody or serum (Table 1). SQMl antibody interfered with epithehal and endothelial cell-cell and cell-ECM adhesion but not with adhesion of fibroblastic and 985

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TABLE 1: Effect of SQMl Antibody on CellAdhesion Cell Type

Monoloay;aF; Type

SW25 scc25 scc25 scC25

scc25 HUVEC fibroblast fibronectin

scC25

colla en HU 43 C

Adherent Cells % of Control 72+5 18~3 94*5

2354

25+5 HUVEC 28+6 HUVEC scC25 20+3 CEM fibronectm 102L5 SCC25= humanheadandneck squamous carcinomacells. HUVEC = humanumbilicalcord vein endothelialcells. CEM = humanlymphoblasticleukemiacells. Resultsare expressedas% of adherentcellsusingcontrol serum or an irrelevant antibody.

lymphocytic cells. The greatest inhibition by SQMl antibody was observed in epithelialendothelial adhesion; moderate inhibition was seen in endothelial-endothelial and epithelial-ECM adhesion; least but significant inhibition was observed in epithelial-epithelial adhesion. Control using normal serum did not result in any inhibition of cell-cell or cell-ECM adhesion. The control antibody, which is known to bind epithelial and endothelial surface membrane, also did not interfere with adhesion. Therefore, antibody binding to the surface membrane was not sufficient, by itself, to block adhesion. These results seem to suggest that SQMl antibody specifically blocked adhesion interactions of epithelial and endothelial cells. Notably, there was greater inhibition of epithelial-endothelial adhesion compared to epithelial-epithelial adhesion, suggesting that the contribution of SQMl protein towards epithelial-endothelial adhesion was greater than in epithelial-epithelial adhesion. Between epithelial cells, there are likely to be many strong adhesion interactions, such as those mediated by desmosomal proteins (13), which are not affected by SQMl antibody. SQMl protein may be more important in mediating epithelial-endothelial and epithelial-ECM interactions, which in turn may be important in the metastastic behavior of epithelial tumors. In Vitro Translation and Immunoblot Analvsis: Before we cloned the SQMl cDNA from the SCC25 lambda gt 11 expression library, immunoblot analysis of the invitro translation products from SCC25 total mRNA was performed to determine whether the epitope could be recognized by the SQMl antibody in a protein made in vitro. As shown in Fig. 1, SQMl antibody recognized two bands (15 and 14 kD) in the SCC25 mRNA translation products (lane 1). Whereas in the control, using rabbit globin mRNA, no such bands were detected (lane 2). Isolation of cDNA of SOMl Protein and Northern Blot Analysis: The number and percentage of recombinants in the SCC25 CDNAlibrarywere 1.0x 105 and 84%, respectively. After repeated screening of approximately 2.5 x 105 phages from the library with SQMl antibody, three candidate clones were found to be strongly reactive. The size of the insert

was approximately other. In Northern

0.5 kb for all three clones and these inserts cross-hybridized with each blot analysis, these cDNA hybridized to a mRNA of size of approximately 986

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kl

kb

< 0.2

02 Immunostainingof in vitro translation productsusing SQMl antibody. E&u SQMl antibody recognizedtwo bandsin the translationproductsof SCC25mRNA (lane 1) but not thoseof globmmRNA (lane 2). Northern blot analysisof SQMl mRNA from squamous carcinomacelllines. lziL2 Lane 1 and lane2 contained5 ug of total RNA from squamous carcinomacell lines,Cam-1 and SK25 respectively.The SQMl probe hybridized to a mRNA of sizeof about 0.6 kb.

600 bp, present in XC25

and Calu-1 cells (Fig. 2). The size of the mRNA

(600 bp) is

compatible with that of the in vitro translation product recognized by SQMl antibody (15 kD). Seauence Analysis: The insert of the cDNA clones was sequencedand the sequence of one of the clone, Q12 is shown in Fig. 3. A polyadenylation signal sequence (14), AAATAAAA

(bps 438 to 445) is located 14 bp upstream of the poly(dA), suggesting

clone Q12 probably contains the complete 3’ end of the gene. The sequence encompassing the first ATG, CAGCCATGG, conforms to the eukaryotic initiation consensus, 987

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-35 CCCTC

BIOCHEMICAL

-30 GGTGCTGCAG

-20 GGATCTGCAG

AND BIOPHYSICAL

-10 GACTGCAGCC

1 ATG

Met 1 CGC TAC Arg Tyr

30 CTG GGC GAT Leu Gly Asp 10

CCG CCA Pro Pro

GAC TAC GGC Asp Tyr Gly 30

45 GCT TCG GTG Ala Ser Val

Glu

TTC Phe

105 CCC AAG Arg Lye

90

135 CAG GAG ATG ATG Gln Glu Met Met

CCC GAA Pro Glu

GAG

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GGG GCG Gly Ala

60 CCC CTG CAG Pro Leu Gln 20

CCC GAC Pro Asp

225 CCA AGT TGC TGG Pro Ser u Trp

CCT GCA Pro Ala

240 255 AGC AGG AAG CGG CAC GAC Ser &a Lys Ara His Asw 80 + + + (+)

270 TCG GGA CTA CTG CGC ACC GCA AGC Ser Gly Leu Leu Ara Thr Ala Ser +

405 GCC CTG TAG GG Ala Leu * 135 460 AGGCCCCTCA

TGC AAA w Lys

420 GTGCACCCCC

470 AAAAAAAAAA

ACC Thr

180 CAC CAC His His 60

Glu

210 TGC AAG CGT GAC AGC TTC Cys Lys Arg Asp Ser Phe 70

360 AGA AGG CGG CAA ATC &rg Ara &q Gln Ile + + + 120

75 TTC Phe

CCA

Pro

TAC TGC GCC Tyr Cys Ala

Glu

150 165 GAC GCG AGT GAG GCT CAG CTG CGG GAC Asp Ala Ser Glu Ala Gln Leu Arg Asp 50

GAG CGG GAC Glx-Arg Asp +

ATG Met

ACA CAG Thr Gln

GAG

195 CTC ATC CGG CTG CTC AAG Leu Ile Arg Leu Leu Lys

300 ATG AAG GAG TTT Met Lvs Glu Phe + 100

CGG Arg

120 ATG GTG GCC Met Val Ala 40

GAG CGC Arg

15 CAC CTG GTC His Leu Val

285 TAT GTG ATG Tvr Val Met

315 330 GAG GGC TGC TCC AGC GGA AGA AGC Glu Glv Cys Ser Ser Glv B;Ta Ser 110 +

CGC Arq +

345 GGC GGG AGA Glv rlv Arg +

375 390 GGC CAG GGA CCC GGG GAA GTG GAC CCC AAG GTG Gly Gln Gly Pro Gly Glu Val Asp Pro Lys Val 130 430 CACCCTATGG

480 AAAAAAAAAA

440 ACCAGTCAAA

450 TAAAACCTTC

486 AAXUA

The DNA sequenceand the deducedaminoacidsequenceof SQMl cDNA. Fie3. The initration consensus se uence, the polyadenylationsignalsequenceand the heptadic repeatof argininesarein bol1 letters. The domaincontainingthe heptadicrepeatof argmines andthe runsof positively-chargedresiduesareunderscored.The positively-chargedresidues within the domain are marked with an “+“. The two cysteinesflanking this domainare double-underscored.

CA(A/G)CCAUGG (15). The first AUG starts an 135 amino acid encoding open reading frame that ends at bp 406, 32 bp upstream of the polyadenylation signal. The calculated molecular weight of the unmodified polypeptide is about 15 kD, which is in close agreement with the size of the in vitro translation products recognized by SQMl antibody (15 and 14 kD). There are three Ser-Gly dipeptides that can form the minimal attachment sites of chondroitin sulfate glycosaminoglycan side chains via 0-glycosyl linkage to serines, as in proteoglycans (16). Proteoglycans are known to interact with ECM and are thought to be involved in cellular adhesion (17). The most striking feature of the deduced polypeptide is the presenceof an almost perfect heptadic repeat of positively-charged arginine residues over a range of 36 residues, near the C-terminal region. This heptadic repeat is found in the region of the polypeptide that can 988

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ro5R+ 98R+ 91 D-

A

B

SQMl Arg His Arg Cys SGS4 SQMl Phe Glu Glu Pro SGS4

85 Asp Glu

Arg Pro 50

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Ser Thr

Gly Glu

Leu Pro 35

Leu Pro

Asp Arg

105 Glu Cys

Gly Glu

Cys Thr

90 Arg Arg

Ser Glu 55

Thr Cys

Ala Glu

Ser Pro

110 Gly Pro

Ser Thr 40

Tyr Glu

Arg Arg

Ser Cys

95 Val Pro

Gly Glu 60

Met Pro

Arg Arg

Gly Thr

115 Arg Thr

Met Cys 45

Lys Glu

Arg Thr

Arg Pro

100 Glu Thr

Arg Arg 65

(A) Heptadicrepeat of argininesin an a-helicalregion of the SQMl polyEi!u peptide. The positively-chargedargininesare alignedon one sideof the helix. (B) Amino acid sequenceof the regionsof SQMl pol eptideandDrosou _hila melanoeaster(Canton-S) Sgs4glue rotein containing the heptaFIC repeat of positively-chargedresidues. The positively-cKargedresiduesare in bold letters.

assume an o-helical secondary structure (18). Within a span of eleven turns of the a-helix, the positively-charged side chains of these six arginine residues are aligned on one side of the o-helix (Fig. 4A). There are only a few sequences in the GenBank/Los Alamos translated sequence database (Release 58) and the National Biomedical Research Foundation (NBRF) protein sequence database (Release 18) containing a heptadic repeat of six or more positively-charged residues. The protein with the highest number of arginines in heptadic repeat is the Drosophila glue protein, Sgs4C1(19), which is used for pupal attachment during metamorphosis (Fig. 4B). It is interesting that, aside from SQMl polypeptide, another protein with a heptadic repeat of positively-charged residues also has adhesive functions. Flanking these heptadic repeats are runs of four positively-charged residues. Charged runs have been found to be important in protein transport, localization and regulatory function (20). In SQMl polypeptide, these runs of positive charges are themselves flanked by cysteines (Fig. 3). These flanking cysteines, through intermolecular or intramolecular disulfide bonds, may stabilize the positions of the runs of positively-charged residues and the aligned positive charges on one side of the helix. This stabilized alignment of positive charges may contribute 989

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A SQHl (107-124) HUMEGFR (219-235) HUMLAP (590-608) HUMGP3A (601-616) HUMCBB (511-528)

CONSENSUS

B HUMLAP SQMl

(653-694) (59-100)

HUMVWFM CONSENSUS

(1624-1638)

C

SGR

+

C

Q

SNNPVKGRTCKERDS-EGCWVAYTLEQQDGMDRYLIYVDESRE .. . . . .:: ::: .:: : . .. . . : AHHLIRLLKCK-RDSFPSCWPASRKRHDSGLLRTASYVMRMKE :: : : ::: : RKEECK-RVSPPSCPP +

CKRS

: :

. :

C

(A) Sequencemotif whichis sharedbetweenSQMl polypeptideand other u humanproteins: EGF receptor (HUMEGFR), p chain of leukocyte adhesionmolecules (HUMLAP), G?IIIa (HUMGP3A), complementc@ (HUMCSB) andvWF (HUI$VWFM). The boxed rest uesare the conservedresiduesamongall sequences. The ammoacrdresidue numbersare in brackets.(B) Sequencesimilarity betweenSQMl 01 e tide, /3 chain of A colon humanleukocyte adhesionproteins(HUMLAP) andhumanvWF (&J?%WM).

denotes identical amino aad residues whereas a period denotes a conservative substitution

of amino acid residues.The aminoacid residuenumbersare in brackets. The consensus sequenceis in bold letters.

to quarternary interaction of SQMl polypeptide with negatively-charged polypeptides. The deduced amino acid sequence of SQMl polypeptide does not reveal any significant hydrophobic region suggestive of a transmembrane domain. However, in light of this possible quarternary interaction, SQMl polypeptide may be stabilized on the surface membrane by its interaction with an integral membrane protein as part of a protein complex. Preliminary immunoprecipitation and immunoblotting studies suggest that SQMl antigen forms complexes with at least two other proteins. Characterization of these other proteins will allow us to clarify the protein-protein interactions of SQMl protein. By comparing with the sequences in the databases, it was found that there were regions in the SQMl polypeptide that were similar to the cysteine repeats of human EGF receptor and to a group of related human adhesion molecules/ligands: /3 subunit of the leukocyte adhesion proteins, endothelial GpIIIa, vWF and complement C8 P chain. The sequence similarity is mainly localized to a sequence motif containing cysteines and a positively-charged residue, found near the C-terminal of the SQMl polypeptide, overlapping two arginines of the heptadic repeat (Fig. 5A). Other than this sequence motif, SQMl polypeptide has an additional region, on the N-terminal side of the heptadic repeat, that is similar to the ~3chain of leukocyte adhesion proteins and vWF (Fig. 5B). The protein most closely similar to SQMl polypeptide is the ~3subunit of the human leukocyte adhesion proteins (21), members of the integrin superfamily. The regions of similarity were located at one of the tandem repeats of the S-cysteine motif and at the proximal extracellular region to the transmembrane domain of the leukocyte adhesion molecules. The region of endothelial GpIIIa, another member of the integrin superfamily, that is similar to SQMl polypeptide is located at one of its cysteine-rich repeat (22). GpIIIais known to complex with GpIIb to form a Ca+ + -dependent heterodimer that can bind fibronectin and vWF (22),

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and can form attachment sites for cytoskeletal proteins (23). Two regions of the SQMl polypeptide are similar to two regions abutting the N-terminus of two of the tandem triple repeats (24) in vWF. It is possible that SQMl polypeptide and vWF are evolutionarily related and the duplication event in vWF occurred subsequent to the divergence of these genes. It is known thatvWF binds to ECM (25) and promotes endothelial cells adhesion (26). The region of complement C8p that is similar to SQMl polypeptide is located near its C-terminus (27). This region is also repeated in its N-terminus as a duplicated sequence. The p chain is known to mediate binding of C8 to C5b-7 during the formation of the membrane pore-forming complex (28). The presented evidence suggests that SQMl polypeptide is related to the P subunit of integrins. However, the limited sequence similarity with the p subunits of integrins, the smaller molecular weight and the lack of a transmembrane domain distinguish it from the integrins. Its presumptive role in cell adhesion of carcinoma cells, endothelial cells and ECM proteins, and its similarity with the leucocyte adhesion molecules and GpIIIa suggest that SQMl polypeptide may play an important role in the metastasis of squamous carcinomas. It is conceivable that these SQMl polypeptide containing squamous carcinoma cells might detach from the primary site and be able to adhere to and penetrate the subendothelial matrix and the endothelium to establish secondary sites. Recently, the elevated expression of a lymphocyte adhesion protein, CDw44 in colon carcinomas has been postulated to facilitate tumor cell adherence to ECM and promote tumor invasiveness (29).

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7. :: 10. ::: 13. 14. 15. 16. 17. 18. ii: 21.

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Fitzgerald, L. A., Steiner, B., Rall, S. C. Jr., and Phillips, D. R. (1987) J. Biol. Chem. 26?,3936-3939. DeJana, E., Languino, L. R., Colella, S., Corbascio, G. C., Plow, E., Ginsberg, M., and Marchisio, P. C. (1988) Blood 71,566-572. Sadler, J. E., SheIton-Inoles, B. B:, Sorace, J. M., Harlan, J. M., Titani, K., and Davie, E. W. (1985) Proc. Natl. Acad. Sa. USA 82,6394-6398. Wagner, D. D., Urban-Pickering, M., and Marder, V. J. (1984) Proc. Natl. Acad. Sci. USA 81,471-475. Dejana, E., Lampugnani, M. G., Giorgi, M., Gaboli, M., Federici, A. B., Ruggeri, Z. M., and Marchisio, P. C. (1989) J. Cell Biol. 109,367-375. Haefliger, J.-A., Tschopp, J., Nardelli, D., Wahli, W., Kocher, H.-P., Tosi, M., and Stanley, K. K. (1987) Biochemistry 26,355 l-3556. Monahan, J. B., and Sodetz, J, M. (1981) J. Biol. Chem. 256,3258-3262. Stamenkovic, I., Amiot, M., Pesando, J. M., and Seed, B. (1989) Cell 56, 1057-1062.

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